Protocol for generating human vascular organoids via orthogonal activation of ETV2 and NKX3.1

Vascular organoids (VOs) are 3D multicellular constructs that model vascular development and function. Here, we present a protocol to generate VOs from human induced pluripotent stem cells via orthogonal activation of transcription factors. We describe steps for inducing endothelial and mural lineages independently using doxycycline-inducible expression of ETV2 and NKX3.1. This protocol enables efficient formation of functional VOs within 5 days, streamlining conventional techniques and supporting applications in vascular biology, tissue engineering, and regenerative medicine. For complete details on the use and execution of this protocol, please refer to Gong et al. 1 • Protocol for generating vascular organoids from human iPSCs • Steps to induce vascular lineages via doxycycline-inducible ETV2 and NKX3.1 • Instructions to embed organoids in extracellular collagen-Matrigel matrices • Guidance on dissociating vascular organoids into single cells for downstream analyses Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Vascular organoids (VOs) are 3D multicellular constructs that model vascular development and function. Here, we present a protocol to generate VOs from human induced pluripotent stem cells via orthogonal activation of transcription factors. We describe steps for inducing endothelial and mural lineages independently using doxycycline-inducible expression of ETV2 and NKX3.1. This protocol enables efficient formation of functional VOs within 5 days, streamlining conventional techniques and supporting applications in vascular biology, tissue engineering, and regenerative medicine.